POS0292 TRANSCRIPTOMIC PROFILING OF SJOGREN’S SALIVARY GLANDS IDENTIFIES FOLLICULAR AND EXTRA-FOLLICULAR GENE SIGNATURES ASSOCIATED WITH RHEUMATOID FACTOR SEROPOSITIVITY
Authors: E. Pontarini, D. Lucchesi, K. Goldmann, M. Lewis, Q. Song, E. Thomas, L. Y. Hao, K. Sivils, C. Pitzalis, M. Bombardieri
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> The presence of circulating rheumatoid factor (RF) and the formation of ectopic lymphoid structures (ELS) in labial salivary glands (SG) of patients with Sjögren's Syndrome (SS) have been reported as independent risk factors associated with the development of SG B-cell MALT lymphoma (MALT-L). Neoplastic MALT-L B-cells express highly hypermutated B-cell receptors bearing RF immunoreactivity in up to 50% of the cases, but whether their maturation and proliferation is dependent on ELS or is also induced by extrafollicular responses in the SG is currently unclear. The definition of ELS and their association with circulating autoantibodies has so far relied on SG histopathology which bears significant limitations. Conversely, molecular pathology analysis through whole-tissue RNA Sequencing (RNASeq) has allowed a better definition of disease heterogeneity and disease taxonomy. <h3>Objectives</h3> To perform transcriptomic profiling of SS minor SG tissue characterised by different degrees of inflammatory aggregate organization and to identify transcriptomic clusters and gene signatures associated with peripheral and histological biomarkers of disease. <h3>Methods</h3> Labial SG were obtained from 99 patients including SS with and without ELS (respectively ELS+ and ELS-, as assessed by immunohistochemistry) and non-specific chronic sialadenitis (Sicca). Total RNA was extracted, complementary DNA libraries were prepared and sequenced. Differentially expressed genes (DEG), deconvolution and pathway analysis were performed. <h3>Results</h3> Unsupervised gene clustering by differential expression between sicca and SS confirmed a clear transcriptome segregation between the two diagnoses. As expected, in SS SG expression of genes associated with inflammation and adaptive immune responses was upregulated (e.g. CCR7, CD19, CR2, CXCL13, CXCL9, CXCR5, FCRL3, FCRL4, IL21R, MS4A1, PAX5, SLAMF6, TLR10) (Figure A). Bulk RNAseq cell deconvolution confirmed immune cell enrichment (Th, B and plasma cells) in SS SG, especially those ELS+. A three-way comparison among sicca, ELS+ and ELS- SG, showed only a few genes specifically associated to ELS- and sicca, whereas most of the DEGs were either ELS+ specific or common to all SS (Figure B). Results of pathway analysis on ELS+ and SS-associated DEGs showed very similar profiles characterised by adaptive and interferon-associated pathway activation. Weighted Gene Co-Expression Network analysis showed higher activation of anti-viral pathways in ELS- SG, where ELS+ SG were enriched in adaptive immune pathways. Principal component analysis on SS SG mRNA showed that a significant proportion of variability was associated with the presence of RF in the serum of patients independently of the presence of ELS. Surprisingly, transcriptome clustering was closer between ELS+/RF+ and ELS-/RF+ SG than the clustering of ELS+/RF- with ELS-/RF- SG. Accordingly, SG of RF+ patients showed significant upregulation of B cell-associated genes (e.g. CXCL13, MS4A1). However, while SG transcriptomes of ELS+/RF+ were characterised by genes associated with germinal centre formation (e.g IL21, AICDA, LTB), transcriptomes from ELS-/RF+ SG displayed a unique set of DEGs such as BCL2, TRIM22, XAF1, DDX58, DDX60 and IFIT1. Conversely, a similar analysis performed based on anti-Ro/SSA and/or anti-La/SSB did not yield any significant differences in gene expression. <h3>Conclusion</h3> A comprehensive bulk RNA sequencing analysis of SS and sicca patients SG showed that higher tissue inflammation with features of functional germinal centres is associated to the presence of both RF and ELS, rather than ELS alone. Furthermore, the existence of a RF-driven SG transcriptome independent of the presence of ELS suggest that both follicular and extra-follicular responses support the selection of B-cells with RF immunoreactivity within the SG of SS patients and could be involved in B-cell lymphomagenesis. <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Elena Pontarini: None declared, Davide Lucchesi: None declared, Katriona Goldmann: None declared, Myles Lewis: None declared, Qingxuan Song Employee of: Janssen Pharmaceuticals, Inc., Spring House, PA, USA, Elizabeth Thomas Employee of: Janssen Pharmaceuticals, Inc., Spring House, PA, USA, Ling-Yang Hao Employee of: Janssen Pharmaceuticals, Inc., Spring House, PA, USA, Kathy Sivils Employee of: Janssen Pharmaceuticals, Inc., Spring House, PA, USA, Costantino Pitzalis: None declared, Michele Bombardieri Speakers bureau: UCB, Janssen, Consultant of: GSK, Janssen, MedImmune, Grant/research support from: Janssen, MedImmune.